Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between fast- and slow-growing king ratsnakes (Elaphe carinata).

In some countries, snakes are important protein sources in human diets, and their economic value depends predominantly on their muscle production, including in the king ratsnake (Elaphe carinata). Muscle growth in the king ratsnake clearly differs among individuals. To date, few potential molecular mechanisms underlying these differences in muscle growth and development have been reported. Here, we integrated mRNA and miRNA expression profiles to screen for genes, pathways, and predicted miRNA-mRNA networks associated with muscle growth and development in fast-growing and slow-growing King ratsnakes. Six hundred eight differentially expressed genes (DEGs) were identified, 48 of which were associated with muscle growth. The 37 genes upregulated in fast-growing individuals (FGIs) may be related to the promotion of muscle growth, whereas the 11 upregulated genes in slow-growing individuals (SGIs) may be related to the inhibition of muscle growth. Seven DEGs were enriched in the PI3K-AKT-MTOR signaling pathway, which appears to promote muscle growth in FGIs. Eleven DEGs were enriched in the ubiquitin-proteasome pathway, which appears to inhibit muscle growth in SGIs. It may interpret why muscle growth differences. Furthermore, 698 miRNA were identified, including 125 novel miRNAs. 63 differentially expressed miRNA (DEMs) were screened, and 950 negative miRNA-mRNA interactions with the 63 DEMs and 608 DEGs were predicted. The miRNA-targeted genes were enriched in pathways related to muscle growth, protein synthesis, and protein degradation. Therefore, in addition to the identified DEGs, miRNAs may play important roles in the differential regulation of muscle growth in FGIs and SGIs of the king ratsnake.

Role of phenotypic plasticity in morphological differentiation between watersnake populations.

An individual's morphology is shaped by the environmental pressures it experiences, and the resulting morphological response is the culmination of both genetic factors and environmental (non-genetic) conditions experienced early in its life (i.e. phenotypic plasticity). The role that phenotypic plasticity plays in shaping phenotypes is important, but evidence for its influence is often mixed. We exposed female neonate diamond-backed watersnakes (Nerodia rhombifer) from populations experiencing different prey-size regimes to different feeding treatments to test the influence of phenotypic plasticity in shaping trophic morphology. We found that snakes in a large-prey treatment from a population frequently encountering large prey exhibited a higher growth rate in body size than individuals in a small-prey treatment from the same population. This pattern was not observed in snakes from a population that regularly encounters small prey. We also found that regardless of treatment, snakes from the small-prey population were smaller at birth than snakes from the large-prey population and remained so throughout the study. These results suggest that the ability to plastically respond to environmental pressures may be population-specific. These results also indicate a genetic predisposition towards larger body sizes in a population where large prey items are more common.

Effects of early nutritional stress on physiology, life histories and their trade-offs in a model ectothermic vertebrate.

Early-life experiences can have far-reaching consequences for phenotypes into adulthood. The effect of early-life experiences on fitness, particularly under adverse conditions, is mediated by resource allocation to particular life-history traits. Reptiles exhibit great variation in life histories (e.g. indeterminate growth), thus selective pressures often mitigate the effects of early-life stress, particularly on growth and maturation. We examined the effects of early-life food restriction on growth, adult body size, physiology and reproduction in the checkered garter snake. Animals were placed on one of two early-life diet treatments: normal diet (approximating ad libitum feeding) or low diet (restricted to 20% of body mass in food weekly). At 15 weeks of age, low-diet animals were switched to the normal-diet treatment. Individuals fed a restricted diet showed reduced growth rates, depressed immunocompetence and a heightened glucocorticoid response. Once food restriction was lifted, animals experiencing nutritional stress early in life (low diet) caught up with the normal-diet group by increasing their growth, and were able to recover from the negative effects of nutritional stress on immune function and physiology. Growth restriction and the subsequent allocation of resources into increasing growth rates, however, had a negative effect on fitness. Mating success was reduced in low-diet males, while low-diet females gave birth to smaller offspring. In addition, although not a direct goal of our study, we found a sex-specific effect of early-life nutritional stress on median age of survival. Our study demonstrates both immediate and long-term effects of nutritional stress on physiology and growth, reproduction, and trade-offs among them.

Facultative mobilization of eggshell calcium promotes embryonic growth in an oviparous snake.

The mineralized eggshell of Reptilia was a major innovation in the evolution of the amniotic egg. Inorganic components strengthen the eggshell and are a potential source of nutrients to developing embryos. Embryos of oviparous reptiles do extract calcium from eggshells but vary interspecifically in exploitation of this resource. The pattern of embryonic calcium nutrition of the corn snake, Pantherophis guttatus, is similar to a diversity of squamate species: embryos obtain most calcium from yolk, yet also mobilize calcium from the eggshell. We tested the hypothesis that embryonic development is not dependent on eggshell calcium by manipulating calcium availability. We peeled away the outer calcareous layer of the eggshell of recently oviposited eggs; control eggs were left intact. Eggs were sampled periodically and calcium content of egg compartments (embryo, yolk, eggshell) was measured. We also analyzed skeletal development and size of hatchlings. There was no difference in survivorship or length of incubation between treatments. However, hatchlings from intact eggs contained more calcium and were larger in mass and length than siblings from peeled eggs. There were no observable differences in ossification but hatchlings from intact eggs had larger skeletal elements (skull, vertebrae). Our results indicate that mobilization of eggshell calcium is not a requirement for embryonic development of P. guttatus and that embryos augment yolk calcium by extracting calcium from the eggshell. This pattern of embryonic calcium nutrition would favor embryos with a greater capacity to mobilize calcium from the eggshell by promoting growth and thereby potentially enhancing hatchling fitness.

Biochemical characterization of venom from Pseudoboa neuwiedii (Neuwied's false boa; Xenodontinae; Pseudoboini).

In this work, we examined the proteolytic and phospholipase A2 (PLA2) activities of venom from the opisthoglyphous colubrid Pseudoboa neuwiedii. Proteolytic activity (3 and 10 µg of venom) was comparable to that of Bothrops neuwiedii venom but less than Bothrops atrox. This activity was inhibited by EDTA and 1,10-phenanthroline but only slightly affected (≤30% inhibition) by PMSF and AEBSF, indicating it was mediated by snake venom metalloproteinases (SVMPs). The pH and temperature optima for proteolytic activity were 8.0 and 37 °C, respectively. The venom had no esterase activity, whereas PLA2 activity was similar to B. atrox, greater than B. neuwiedii but less than B. jararacussu. SDS-PAGE revealed venom proteins >100 kDa, 45-70 kDa, 21-24 kDa and ~15 kDa, and mass spectrometry of protein bands revealed SVMPs, cysteine-rich secretory proteins (CRISPs) and PLA2, but no serine proteinases. In gelatin zymography, the most active bands occurred at 65-68 kDa (seen with 0.05-0.25 µg of venom). Caseinolytic activity occurred at 50-66 kDa and was generally weaker than gelatinolytic activity. RP-HPLC of venom yielded 15 peaks, five of which showed gelatinolytic activity; peak 7 was the most active and apparently contained a P-III class SVMP. The venom showed α-fibrinogenase activity, without affecting the ß and γ chains; this activity was inhibited by EDTA and 1,10-phenanthroline. The venom did not clot rat citrated plasma but reduced the rate and extent of coagulation after plasma recalcification. In conclusion, P. neuwiedii venom is highly proteolytic and could potentially affect coagulation in vivo by degrading fibrinogen via SVMPs.

Immune configuration in hatchling snakes is affected by incubation moisture, and is linked to subsequent growth and survival in the field.

We incubated the eggs of field-caught keelback snakes (Tropidonophis mairii) on wet versus dry substrates to explore impacts of incubation conditions on white blood cell (WBC) concentrations and differential WBC counts of hatchlings. In a second, independent study young snakes were released into the field, allowing us to explore fitness correlates of WBC profiles. Dry incubation reduced embryonic survival and hatchling body size, thus decoupling egg size from hatchling size. Incubation conditions also altered WBC profiles. Lymphocyte and azurophil counts were related to hatchling body size but not to initial egg mass, whereas heterophil counts were related to both of these traits. The egg-size effect on heterophil counts may reflect a maternal effect on offspring immune configuration. The ratio of heterophils to lymphocytes (an index of stress) was higher in hatchlings from eggs incubated on dry substrates. Snakes with higher counts of lymphocytes at hatching were more likely to be recaptured as adults (H:L; an index of survival), whereas snakes with higher basophil counts exhibited more rapid growth. In summary, our experiments show that incubation moisture levels influence the immune configuration of hatchling snakes, and that variation among individuals in WBC counts at hatching is a significant predictor of an individual's fitness after it is released into the wild. The demonstrated link between incubation conditions and offspring fitness is likely to impose strong selection on maternal nest-site choice.

Extra-Mediterranean glacial refuges in barred and common grass snakes (Natrix helvetica, N. natrix).

Extra-Mediterranean glacial refugia of thermophilic biota, in particular in northern latitudes, are controversial. In the present study we provide genetic evidence for extra-Mediterranean refugia in two species of grass snake. The refuge of a widely distributed western European lineage of the barred grass snake (Natrix helvetica) was most likely located in southern France, outside the classical refuges in the southern European peninsulas. One genetic lineage of the common grass snake (N. natrix), distributed in Scandinavia, Central Europe and the Balkan Peninsula, had two distinct glacial refuges. We show that one was located in the southern Balkan Peninsula. However, Central Europe and Scandinavia were not colonized from there, but from a second refuge in Central Europe. This refuge was located in between the northern ice sheet and the Alpine glaciers of the last glaciation and most likely in a permafrost region. Another co-distributed genetic lineage of N. natrix, now massively hybridizing with the aforementioned lineage, survived the last glaciation in a structured refuge in the southern Balkan Peninsula, according to the idea of 'refugia-within-refugia'. It reached Central Europe only very recently. This study reports for the first time the glacial survival of a thermophilic egg-laying reptile species in Central Europe.

Physiological and behavioral responses to salinity in coastal Dice snakes.

Secondarily marine tetrapods have evolved adaptations to maintain their osmotic balance in a hyperosmotic environment. During the transition to a marine habitat, the evolution of a euryhaline physiology likely encompassed successive changes in behavior and physiology that released organisms from regular access to fresh water. Deciphering these key steps is a complicated task. In this study, we investigated a species of freshwater natricine snake in which some populations are known to use marine environments. We experimentally subjected 30 adult Dice snakes (Natrix tessellata) from a population inhabiting the Black Sea coast to three salinities corresponding to freshwater (~0.1‰), brackish water (~15.0‰), and full-strength seawater (~34.0‰) in order to investigate their physiological (variation of body mass, osmolality) and behavioral (activity, drinking behavior) responses to salinity. Our results show that coastal Dice snakes from the study population are relatively tolerant to salinity close to that recorded in the Black Sea, but that prolonged exposure to full-strength seawater increases osmolality, stimulates thirst, decreases the activity of snakes and may ultimately jeopardize survival. Collectively with previously published data, our results strongly suggest specific physiological adaptations to withstand hyperosmolality rather than to reduce intake of salt, in coastal populations or species of semi-aquatic snakes. Future comparative investigations of Dice snakes from populations restricted to freshwater environment might reveal the functional traits and the behavioral and physiological responses of coastal N. tessellata to life in water with elevated salinity.

A new species of Rhadinella (Serpentes: Colubridae) from the Sierra Madre del Sur of Guerrero, Mexico.

We describe a new species of Rhadinella from the Sierra Madre del Sur of Guerrero, Mexico, a region where the genus was previously unknown. This diminutive species is a member of a group of snakes previously allocated in the Rhadinaea godmani group, and more recently transferred to the genus Rhadinella. These snakes may have conspicuous dark longitudinal striping on a pale brown to orange background or may have dark brown to blackish dorsal ground coloration, which mostly or completely obfuscates a pattern of longitudinal striping. The new species is mostly dark with barely discernible slightly paler or darker striping (depending on how striping is interpreted). The closest relative of the new species, on the basis of morphological similarities and biogeography, appears to be Rhadinella donaji which occurs to the east in the Sierra Madre del Sur of Oaxaca about 275 km from the type-locality of the new species.

Resurrection of Pseudorabdion torquatum (A.M.C. Duméril, Bibron & A.H.A. Duméril, 1854), a former synonym of P. longiceps (Cantor, 1847) (Colubridae: Calamariinae) from Sulawesi, Indonesia.

The colubrid snake species Pseudorabdion torquatum is resurrected from the synonymy of P. longiceps. Out of the three syntypes, only two were located and examined at Muséum national d'Histoire naturelle, Paris, France (MNHN-RA). Pseudorabdion torquatum differs from P. longiceps in having a larger SVL (227.0 mm vs. 132.0-193.0 mm in males); higher number of ventral in males, 141 (vs. 127-139); lower number of ventrals in females, 137 (vs. 139-145); lower number of subcaudals in both sexes, 12-17 (vs. 17-30); and six supralabials (vs. five). Pseudorabdion torquatum is thus regarded as a valid and endemic species to Sulawesi. The species is here redescribed and a lectotype is designated. Furthermore we provide an English translation of the original description of Rabdion torquatum which was originally written in French. Thus, besides unconfirmed records of P. longiceps from Sulawesi, there is a single known congener from that island, P. sarasinorum.

A new species of kukri snake (Squamata: Colubridae: Oligodon Fitzinger, 1826) from Con Dao Islands, southern Vietnam.

We describe a new kukri snake, Oligodon condaoensis sp. nov., from Con Dao Islands, southern Vietnam based on the morphological characters of four specimens. It differs from other congeners by a combination of the following characters: medium size in adults (total length up to 552 mm); 17-17-15 dorsal scale rows; deeply forked hemipenes without spines and papillae, extending to subcaudal 13 or 14; 11-13 maxillary teeth, the posterior three being enlarged; cloacal plate undivided; head scalation complete; nasal divided; presubocular absent; 168-176 ventrals; 33-37 subcaudals; overall dorsal coloration dark gray, faint body stripes present or absent; and ventral coloration cream to dark gray without rectangular blotches. We also provide a list of 43 species of amphibians and terrestrial reptiles recorded from Con Dao Islands.

Insulin-like signaling (IIS) responses to temperature, genetic background, and growth variation in garter snakes with divergent life histories.

The insulin/insulin-like signaling pathway (IIS) has been shown to mediate life history trade-offs in mammalian model organisms, but the function of this pathway in wild and non-mammalian organisms is understudied. Populations of western terrestrial garter snakes (Thamnophis elegans) around Eagle Lake, California, have evolved variation in growth and maturation rates, mortality senescence rates, and annual reproductive output that partition into two ecotypes: "fast-living" and "slow-living". Thus, genes associated with the IIS network are good candidates for investigating the mechanisms underlying ecological divergence in this system. We reared neonates from each ecotype for 1.5years under two thermal treatments. We then used qPCR to compare mRNA expression levels in three tissue types (brain, liver, skeletal muscle) for four genes (igf1, igf2, igf1r, igf2r), and we used radioimmunoassay to measure plasma IGF-1 and IGF-2 protein levels. Our results show that, in contrast to most mammalian model systems, igf2 mRNA and protein levels exceed those of igf1 and suggest an important role for igf2 in postnatal growth in reptiles. Thermal rearing treatment and recent growth had greater impacts on IGF levels than genetic background (i.e., ecotype), and the two ecotypes responded similarly. This suggests that observed ecotypic differences in field measures of IGFs may more strongly reflect plastic responses in different environments than evolutionary divergence. Future analyses of additional components of the IIS pathway and sequence divergence between the ecotypes will further illuminate how environmental and genetic factors influence the endocrine system and its role in mediating life history trade-offs.

Pigment cell mechanism of postembryonic stripe pattern formation in the Japanese four-lined snake.

Postembryonic changes in the dermal and epidermal pigment cell architecture of the striped and nonstriped morph of the Japanese four-lined snake Elaphe quadrivirgata were examined to reveal stripe pattern formation after hatching. The striped and nonstriped morphs were distinguishable at the hatching, suggesting that the basis of stripe pattern was formed during embryonic development. In the striped morph, the color of stripes changed from red-brown in juveniles to vivid dark-brown in adults, and density of dermal melanophore increased much more in the stripe than background dorsal scales with growth. This increase in density of dermal melanophore was accompanied not only by the increased epidermal melanophore density but also by the change in vertical structures of dermal melanophore. By contrast, the density of epidermal and dermal melanophore evenly increased over the dorsal scales in the nonstriped morph. Thus, the increased vividness of the stripe pattern after hatching is achieved through localized increase of melanophore density particularly in the stripe region but not over the whole dorsal scales.

Taxonomic status and morphological variation of Hydrodynastes bicinctus (Hermann, 1804) (Serpentes: Dipsadidae).

Hydrodynastes bicinctus was described with no type material or locality and it has two subspecies currently recognized that are not taxonomically well defined. We tested the validity of the two subspecies through meristic, morphometric, and color pattern characters. Two apparently distinct color patterns of H. bicinctus were noticed, one from the Cerrado open formations and the other from the Amazon rainforest. These aforementioned patterns, however, exhibited a high degree of geographic overlap and many specimens showed a blended pattern. Based on these results we propose synonymizing H. bicinctus schultzi with the nominal taxon. Furthermore, we designate a neotype for the species, present data on geographic distribution, and provide morphological descriptions of the hemipenis, cephalic glands, and skull.

Taxonomic revision of Chironius flavolineatus (Jan, 1863) with description of a new species (Serpentes: Colubridae).

We conducted a taxonomic review of Chironius flavolineatus on the basis of continuous and discrete morphological characters. We recognize a new species which is distinguished from all currently recognized congeners by the following unique combination of characters: first third of body black or dark gray; vertebral stripe yellowish or creamish white distinct from dorsals of nape and extending throughout almost whole body length; head dorsum tan to brown, distinct from background color of first third of body; posterior temporal scales ranging one to four; cloacal shield frequently divided; two to four rows of keeled dorsal scales at midbody; venter ground color gradually darkening towards cloaca; region of medial constriction of hemipenis slightly covered with spinules separating calyces of apex from spines below region of constriction; in lateral view, sulcus spermaticus positioned on convex face of hemipenis; ascending process of premaxilla oblique anteroposteriorly to longitudinal axis of skull; optic fenestrae not exceeding frontoparietal suture; posterior border of supratemporal exceeding braincase; dorsoventral axis of quadrate oblique mesolaterally, moving away from longitudinal axis of skull. Furthermore, we provide data on morphological variation, distribution, and an emended diagnosis for C. flavolineatus.

A new species of the genus Oligodon Fitzinger, 1826 (Squamata: Colubridae) from coastal southern Vietnam.

A new species of the genus Oligodon from the coastal area of Binh Chau-Phuoc Buu Nature Reserve, Ba Ria-Vung Tau Province, southern Vietnam, is described. Oligodon arenarius sp. nov. is distinguishable from all other species by the unique combination of the following characters: medium size; 17 dorsal scale rows; 6-8 maxillary teeth, the posterior three being enlarged; head scalation lacking a loreal but usually including a presubocular; divided nasal; two postoculars; 131-144 ventrals; 36-60 subcaudals; unforked hemipenis, without spines or obvious papillae; sexual dimorphism displayed in the number of subcaudals (36-40 in females, 58-60 in males) and a relative tail length, tails being quite long in males (TaL/TL = 0.26-0.28) and moderate in females (TaL/TL = 0.13-0.17); head coloration pattern including ocular band, temporal bands and chevron-shaped mark on nape; dorsal coloration without distinct pattern, uniform or with dark speckling; ventrals pinkish in life, immaculate. To date, this species appear to be endemic to Vietnam.

Genetic differentiation among species of the genus Thermophis Malnate (Serpentes, Colubridae) and comments on T. shangrila.

The genus Thermophis includes the two species, T. baileyi and T. zhaoermii, which differ morphologically, geographically and molecularly. Recently, a third Thermophis species was described from Shangri-La, northern Yunnan Province, China, and named T. shangrila. The new species was based on morphological and genetic data derived from three specimens. However, the morphological features used to delimit this species seem vague, because they may fall within the range of intraspecific variation of T. zhaoermii. Furthermore, the reported genetic differences in nuclear data are questionable. They likely resulted from a misinterpretation probably due to alignment/analytical flaws or sample/sequence mix-up. Here, we used partial sequences of three mitochondrial (CO1, ND4, cytb) genes and one nuclear (c-mos) gene to analyse the genetic variation between and within species of Thermophis. We inferred the phylogeny using Bayesian Inference and Maximum Likelihood approaches and present additional morphological data that contribute to the knowledge on intraspecific variation in the genus. Our results indicate lacking robustness in the distinguishing morphological features and in the genetic differentiation of T. shangrila and highlight the need for more detailed morphological and molecular studies from a substantially larger sample.

Molecular phylogeny of Caribbean dipsadid (Xenodontinae: Alsophiini) snakes, including identification of the first record from the Cay Sal Bank, The Bahamas.

We document the first specimen of a dipsadid snake from the Anguilla Cays, Cay Sal Bank, The Bahamas. We analyze 3,426 base pairs (bp) of sequence data derived from five mitochondrial loci and one nuclear locus using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Our molecular data agree with some aspects of morphology (e.g., scale counts, dentition, and color pattern) supporting identification of this specimen as the Cuban Racer, Cubophis cantherigerus cantherigerus (Bibron 1840), a species previously regarded as endemic to Cuba. This discovery provides another example of the strong Cuban affinities of the terrestrial vertebrate fauna of Bahamian islands.

Phylogeny of the genus Rhynchocalamus (Reptilia; Colubridae) with a first record from the Sultanate of Oman.

The genus Rhynchocalamus comprises three species distributed in Southwest Asia. Little is known about them, most probably because of their secretive fossorial lifestyle. The poor knowledge of the genus is even underscored by the fact that its phylogenetic affinities remained unclear until very recently. The least known of the species, Rhynchocalamus arabicus, is known only from the holotype collected in Aden, Yemen, and it has not been observed since its description in 1933. Here we provide a second record for this species, which represents the first record of this genus for Oman. This extends its range in southern Arabia by more than 1000 km. The observed specimen was determined as R. arabicus on the basis of its similarity in size, color, and scalation with the holotype. Furthermore, we sequenced three mitochondrial (12S, 16S, cytb) and one nuclear (cmos) genes for R. arabicus and for two individuals of R. melanocephalus and one R. satunini and inferred the phylogenetic relationships of all currently recognized species of the genus for the first time. The results of our phylogenetic analyses indicate that Rhynchocalamus is a member of the Western Palearctic clade of Colubrinae and is sister to Lytorhynchus, with which it forms a very well supported clade and shares some morphological characters. As our results show, R. satunini is the basal lineage of the genus and R. melanocephalus is sister to R. arabicus.